Th1 and Th17 cells specific for neuroantigens such as myelin oligodendroglial glycoprotein (MOG), myelin basic protein (MBP) and proteolipid protein (PLP) can induce severe signs of experimental autoimmune encephalomyelitis (EAE) in mice that include ascending paralysis, demyelination and axonal damage. These T cell specificities are present and may contribute to central nervous system (CNS) damage in subjects with multiple sclerosis (MS). Our laboratory designed and developed a new class of antigen specific and highly potent minimal recombinant T cell receptor (TCR) ligands (RTL) that can both prevent and reverse clinical and histological signs of EAE after onset of EAE. RTL constructs are comprised of a single exon that includes covalently linked 11 domains of MHC class II (a partial MHC moiety) with an attached antigenic peptide. RTL are highly stable proteins of ~25kD produced in E. coli. Upon purification and folding, RTL assume a conformation similar to the distal domains of full-length class II molecules that bind and present antigenic peptides to activate cognate T cells. RTL1000, comprised of the human MOG-35-55 peptide covalently linked to the MS risk factor, HLA-DR2b (DRB1*1501), is currently being tested in a Phase 1 clinical trial as a novel therapy for MS. Although our studies have documented the ability of RTL to deliver inhibitory signals through the TCR of cognate T cells in a peptide-specific manner, new data showing rapid antigen non-specific RTL binding to antigen-presenting cells (APC), including macrophages, dendritic cells (DC) and B cells, strongly suggest alternative tolerogenic pathways. Both mice and humans have readily detectable levels of natural IgM and IgG antibodies specific for RTL that would likely bind to and signal through Fc receptors (FcR) when complexed with injected RTL, and it is possible that this pathway involves inhibitory FcR such as Fc?RIIB. However, B cell knockout mice with EAE can still be fully treated with RTL in the absence of anti-RTL antibodies, indicating that B cells and Ab mediated signaling alone are not sufficient to account for the therapeutic effects of RTL. RTL binding to APC is not inhibited by Fc-Blocker, suggesting a third pathway that involves RTL binding to unique RTL-specific receptors (RTLR), a possibility that is now well supported by inhibition of passive EAE using RTL-armed APC and the demonstration of high affinity RTLR on splenic APC and candidate RTLR proteins. These new findings support our general hypothesis that RTL-induced regulation of antigen specific T cells involves at least 3 separate and possibly redundant inhibitory pathways. RTL binding to FcR or unique RTL receptors has profound implications in that the RTL binding event itself may activate crucial tolerogenic pathways in APC that may ultimately deliver an inhibitory signal to the antigen- specific T cell. Because we have not yet demonstrated a role for FcR binding of RTL:Ab complexes in the RTL therapeutic mechanism, we here propose to focus our attention on T cell tolerance induction by direct RTL ligation of the cognate TCR and by TCR interactions induced by RTL-armed APC. Aim 1. Assess effects of direct RTL ligation of TCR on Ag specific T cells. Early and downstream activation events and kinetics of binding with TCR will be evaluated in MBP-, PLP- and MOG-specific encephalitogenic T cells ligated with free cognate and control RTL. T cells pretreated with RTL prior to activation with APC/peptide will be evaluated for TCR signaling changes, cytokine profiles and ability to transfer EAE into naive recipient mice. Aim 2. Evaluate TCR signaling after RTL binding to an RTL receptor (RTLR) on APC. RTL treatment of EAE will be evaluated after blocking cell binding with RTL-specific Fab. Early and downstream T cell activation will be evaluated after ligation of the TCR with cell bound cognate and control RTL. APC from RTL-treated mice will be evaluated ex vivo for ability to induce specific tolerance in indicator T cells with cognate TCR. The RTLR will be sequenced and characterized. Our studies will provide unique insights into the mechanisms of RTL therapy for EAE in mice, with implications for similar evaluations in subjects with MS.